Stem cell gene therapy using viral vectors has two types of unwarranted effects: decreased efficacy caused by the silencing of the vectors due to inactivation by the surrounding heterochromatin; or genotoxicity caused by activation by the vector's enhancers of deleterious genes in the vicinity of the vector. It is generally assumed that these problems can be avoided by the use of chromatin insulators. Currently there is limited knowledge about chromatin insulators in general and insulators of the mammalian and human genome in particular. The goal of this project is first, to use the genomic approaches our group has pioneered to identify elements of the human genome that have the potential to act as chromatin insulators and second, validate their insulator activity with functional assays. Specifically: 1) We will use a high resolution genomic chromatin immunoprecipitation approach. Chip sequencing, to identify all of the binding sites for two proteins known to be associated with chromatin insulators, CTCF and USF1/2, in the genomes of cell lines and primary cells included in our databases. 2) Under the hypothesis that chromatin insulators present themselves as DNAse I hypersensitive sites (DHSs) placed between active chromatin domains, we will use genomic high throughput approaches to identify as potential constitutive chromatin insulators those intergenic DHSs which are present in the chromatin profiles of all cell lines and primary cell lineages of our databases. We will also test the hypothesis of lineage-specific chromatin insulators by profiling the DHSs of primary human fetal and adult erythroid cells and identifying as potential insulators those erythoid lineage-specific DHSs which are located in the borders of active erythroid gene domains. We will then informafically compare the constitutive and erythroid-specific DHS profiles with the CTCF and USF1/2 profiles in order to identify genomic elements that exhibit all three features and other genomic features that may be exhibited by chromatin insulators, such as specific histone modifications and genomic boundaries. 3) We will validate the insulator function of the so identified elements with functional enhancer-blocking, chromatin barrier and genotoxicity prevention assays. It is expected that these studies will identify new potent insulator elements for use in gene therapy vectors and will also provide new information about the organization and function of the chromatin insulators in the human genome.